Abstract (inglese)

Crohn's disease is a chronic inflammatory disease that can affect any segment of the intestinal tract, with a peak of incidence between 25 and 35 years of age affecting 2-7/100000 people per year.
More than 33% of patients suffering from Crohn's disease develop intestinal stenosis, preferentially in the small intestine, within 10 years of diagnosis and the only treatment available is surgery. Recurrences occur in 75% of cases.
The repairing process of an organ after a chronic damage, like Crohn's disease, is a delicate balance between the production of new extracellular matrix and destruction of the excess. When this balance is lost, the physiological process of repair leads to fibrosis characterized by overgrowth and scarring of tissue with the loss of normal functions. Sub-epithelial myofibroblasts are the main cells involved in deposition of extracellular matrix in the bowel and their role is now under investigation by many groups looking at several pathogenetic processes such as inflammation and cancer.
TAK1 is a MAP3K activated by mitogen and inflammatory stimuli and involved in various intracellular signalling cascades. Among several cytokines able to activate TAK1 there is also TGF-?, a known profibrogenic cytokine, that shows high expression in the tissue affected by excessive fibrosis in Crohn's disease. Recently, the role of TAK1 has been associated with cardiac hypertrophy and renal fibrosis, this prompted us to investigate its role in the fibrogenic process in Crohn's disease.
To study the involvement of TAK1 in fibrogenetic Crohn's disease we initially determined the expression levels and phosphorylation status of the protein in the ileal mucosa obtained from patients undergoing surgical resection for stenosis and in normal ileum specimens obtained from patients undergoing colectomy for cancer. The data obtained through western blotting analysis showed an increase in TAK1 expression in fibrotic tissue associated to an increased phosphorylation state, which characterizes its activation.
Immunohistochemical studies, through co-localization with ?SMA, demonstrated a massive presence of myofibroblasts in the mucosa of patients with Crohn's disease, that expressed TAK1 and its phosphorylated form, mainly in the periglandular region.
The functional characterization of TAK1 was therefore performed on primary ISEMFs extracted from regions of Crohn’s associated fibrosis and normal ileum. Cells derived from fibrotic tissues maintained the disease phenotype in vitro and showed higher levels of TAK1 expression and greater activated status compared to control cells. By immunocytochemical and western blotting studies we observed that TGF-? induced TAK1 phosphorylation in intestinal myofibroblasts.
For this reason we started functional studies of gene silencing by RNA interference using an adenoviral vector. Collagen 1? mRNA was analyzed by Real Time PCR in ISEMF after TGF-? stimulus or silencing with the viral vector. The expression of collagen was higher in cells derived from Crohn’s disease specimens than in controls; however TGF-? induced more collagen expression in control cells. Tak1 silenced cells showed significantly reduced basal collagen expression and prevented TGF-?-induced collagen up-regulation. Using a TAK1 inhibitor we confirmed the data obtained with siRNA at both the RNA level and the protein level. In addition, myofibroblasts treated with TAK1 inhibitor or siRNA showed a reduced migratory capacity in a wound healing assay.
To assess whether TAK1 can be used as an early marker of fibrogenetic process in Crohn's disease, we correlated its expression in a population of patients with Crohn's disease in remission and without clinical signs of stenosis, with markers of fibrosis. Preliminary data showed that the increased expression of Tak1 in Crohn’s mucosa correlated with the expression of two important fibrotic markers, collagen 1? and ?SMA.
In conclusion, the results of this study support the role of TAK1 in intestinal myofibroblasts during the TGF-? -induced fibrogenesis process in Crohn’s disease and further studies might indicate whether it is a suitable therapeutic target as well as a prognostic tool.

Gasche C, Scholmerich J. et al A simple classification of Crohn’s disease: Reporto of the working Party for the World Congresses of Gastroenterology, Vienna 1988.Inflamm Bowel Dis 2000;6:8-15
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